Chimeric polypeptide comprising the fragment b of shiga toxin and peptides of therapeutic interest

ABSTRACT

The invention pertains to methods for using chimeric polypeptides of the formula: 
       B-X 
     wherein B represents the B fragment of Shiga toxin or a functional equivalent thereof, and X represents one or more polypeptides of therapeutic significance. Compositions for therapeutic use comprising the polypeptide B-X are also included.

This application is a divisional of U.S. patent application Ser. No.10/443,614, filed May 21, 2003, which is a divisional of U.S. patentapplication Ser. No. 09/484,471, filed Jan. 18, 2000, now U.S. Pat. No.6,613,882, which is a continuation of PCT/FR98/01573, filed Jul. 17,1998, which claims priority to French Patent Application No. 9709185,filed Jul. 18, 1997. The entire contents of each being incorporatedherein by reference in their entirety.

The invention relates to means and to their use for intracellulartransport of proteins or polypeptides, also to the membrane presentationof certain epitopes.

Retrograde transport can be defined as the movement of molecules fromthe cell membrane to the endoplasmic reticulum (ER), passing ifnecessary via the Golgi apparatus. This mechanism has been demonstratedfor certain classes of proteins of the endoplasmic reticulum carryingthe tetrapeptide KDEL at the carboxy terminal (or HDEL in starch). Agreat deal of biochemical and morphological evidence indicates thatthose proteins leave the endoplasmic reticulum, reach the Golgiapparatus in which modifications are made to their carbohydrate chainand are then redirected to the endoplasmic reticulum. The tetrapeptideKDEL is a retention signal which traps the peptide or protein to whichit is attached in the endoplasmic reticulum, such trapping taking placeby interaction at a receptor protein for the KDEL motif described byLewis M. J. et al in Nature, 348 (6297): 162: 3, 1990, 8^(th) Nov.

Other evidence for the existence of intracellular retrograde transportarises from a study of certain bacterial toxins which enter the cytosolof eukaryotic cells after passing into the endoplasmic reticulum (Pelhamet al (1992) Trends cell. Biol., 2: 183-185). A particular example whichhas been studied is that of the Shiga toxin from Shigella dysenteriae,also E. coli Shiga-like toxins. Such toxins are composed of twopolypeptide chains; one (the A fragment) is the toxic fragment andcarries a deadenylase activity which inhibits protein synthesis byacting on the 28S ribosomal RNA, while the other sub-unit (the Bfragment) enables the toxin to bind to the target (O'Brien et al (1992),Curr. Top. Microbiol. Immunol. 180: 65-94). Electron microscope studieshave shown that Shiga toxin can be detected in the ER of A431, Vero, andDaudi cells in particular (Sandvig et al 1992 and 1994; KHINE, 1994).Further, treating cells with a fungal metabolite which cause the loss ofthe Golgi apparatus structure (brefeldin A) protects the cells againstShiga toxin thus suggesting that they traverse the Golgi apparatusbefore reaching the ER. Finally, Kim et al (1996) have confirmed thatthe B fragment of the toxin is localised in the Golgi apparatus.

The following references demonstrate the state of the art as regardsretrograde transport, in particular transport of the B fragment of Shigatoxin in the ER: Sandvig et al (1992), Nature 358:510-512; Sandvig et al(1994) J Cell. Biol 126: 53-64; Kim et at (1996) J. Cell. Biol 134:1387-1399.

Intracellular transport is defined as the ensemble of exchanges betweenthe different cellular compartments.

The authors of the present application have observed that the B fragmentis not only moved towards the ER, but also to the nucleus ofhematopoietic lines, in particular dendritic cells and macrophages.

The authors have shown that those cells, incubated in the presence oftwo micromoles of B-Gly-KDEL fragment, as described below, for 3 hoursthen fixed, have a reactivity with specific antibodies against the toxinin the nucleus and even in the nucleole of such cells (unpublishedresults) which clearly indicate the existence of intracellular transportof that fragment.

The present invention results from observations on intracellulartransport of the B fragment of Shiga toxin (B fragment) and uses itsrouting properties to construct a chimeric polypeptide sequencecontaining:

-   -   either a peptide or a polypeptide of therapeutic significance        bound to said fragment or any functional equivalent thereof;    -   or a polynucleotide sequence carrying a sequence the expression        of which is desired. The B fragment and the polynucleotide        sequence are coupled using any technique which is known to the        skilled person and in particular that described by Allinquant B.        et al in the Journal of Cell Biology 1288 (5): 919-27 (1995).

In addition to covalent coupling of DNA molecules or other molecules tothe B fragment, coupling can be via a strong non covalent interaction.To this end and by way of example, the cDNA of the B fragment is fusedwith that of streptavidin or with any other avidin derivative usingknown methods (Johannes et al (1987), J. Biol. Chem., 272: 19554-19561).

The protein resulting from fusion (B-streptavidin) can react withbiotinylated DNA obtained by PCR using biotinylated primers, or with anyother biotinylated substance. The resulting complex is bound to targetcells and should be transported like the intracellular B fragment.

A further coupling method employs site-specific biotinylation of the Bfragment. To this end, the cDNA of the B fragment is fused with cDNAcoding for the BirA enzyme recognition site Boer et al (1995), J.Bacterial, 177: 2572-2575; Saou et al (1996) Gene, 169: 59-64). After invitro biotinylation, the B fragment is bound to other biotinylatedmolecules (such as cDNA, see above) via streptavidin or any othertetravalent avidine derivative.

The term “functional equivalent” means any sequence derived from the Bfragment by mutation, deletion or addition, and with the same routingproperties as the B fragment.

More precisely, a functional equivalent can be constituted by anyfragment with the same retrograde transport properties and evenintracellular transport to the nucleus as those described for the Bfragment. Examples which can be cited are the B fragment of verotoxindescribed in the Proceedings of the National Academy of Sciences of theUnited States of America, 84 (13): 4364-8 1987, Jul., or the B fragmentfrom ricin described by Lamb F. I. Et al in the European Journal ofBiochemistry, 148(2): 265-70 (1995). After describing the particulartransport properties of such fragments, the skilled person will be ableto select the fragment which would be the best candidate as a vector forrouting any sequence in any cellular compartment.

Thus the present invention encompasses the use of the B fragment ofShiga toxin or any other sub unit of bacterial toxins which would havecomparable activities, in particular routing properties analogous tothose of fragment B, including polypeptides miming the Shiga toxin Bfragment. These polypeptides, and in general these functionalequivalents, can be identified by screening methods which have in commonthe principle of detecting the interaction between random peptidesequences and the Gb₃ receptor or soluble analogues of the receptor. Byway of example, phage libraries expressing random peptide sequences forselection on affinity columns comprising Gb₃ or after hybridisation withsoluble radioactive Gb₃ analogues can be used. The glycolopid Gb₃ hasbeen identified as being the cellular receptor of the Shiga toxin(Lingwood (1993), Adv. Lipid Res., 25: 189-211). Gb₃ is expressed bycells which are sensitive to the toxin and internalisation of the toxinwould be permitted by an interaction with Gb₃. The present inventorshave demonstrated that in HeLa cells in which expression of the Gb₃receptor has been inhibited (FIG. 1A), the intemalised B fragment is nottransported into the Golgi apparatus but is accumulated in vesicularstructures in the cytoplasm, principally represented by lysosomes. Inthe control cells, the B fragment is transported to the Golgi apparatus(FIG. 1B).

This hypothesis, whereby in the absence of the Gb₃ receptor, the Bfragment is no longer transported to the biosynthesis system orsecretion system, has been confirmed by biochemical experiments (FIG.2).

The inventors have demonstrated that in the presence of an inhibitor ofGb₃ receptor synthesis, PPNP (+PPNP), up to 50% of the intemalised Bfragment is degraded in the form of TCA-soluble material, which conformsto a transport activity towards a subsequent degradation compartmentsuch as an endosomal or lysosomal compartment. When Gb₃ receptorsynthesis is not inhibited (−PPNP), a much smaller proportion ofinternalised B fragment becomes TCA soluble. It can thus be concludedthat the presence of the Gb₃ receptor is necessary for addressing the Bfragment to specific compartments, which tends to favour the fact themain factor in the activity of the B fragment is its binding to the Gb₃receptor.

The present invention provides chimeric polypeptide sequences, saidsequences comprising at least: the Shiga toxin B fragment or afunctional equivalent thereof the carboxy-terminal end of which hasbound to it one or more X polypeptides with the following formula:

B-X,

wherein:

-   -   B represents the B fragment of a toxin such as the Shiga toxin,        the sequence of which has been described by N. G. Seidah et al        (1986), J. Biol. Chem. 261: 13928-31, and in Strockbine et al        (1988), J. Bact. 170: 1116-22, or a functional equivalent        thereof, or from verotoxin or from ricin (references supra);    -   X represents one or more polypeptides the upper limit to the        total length of which being that of compatibility with        retrograde or intracellular transport.

The present invention also provides chimeric molecules with thefollowing structure:

B-X′

where B has the same meaning as above and X′ represents a nucleotidesequence coding for a peptide sequence X the expression of which isdesired, in particular an antigen epitope.

The chimeric molecules of the invention can also comprise:

-   -   a) modification sites such as an N-glycosylation site        constituted by about 20 amino acids, phosphorylation sites or        any sequence necessary for any maturation of the molecule;    -   b) a retention signal of the tetrapeptide KDEL type        (Lys-Asp-Glu-Leu) which,        when it is bound to the carboxy-terminal end of resident ER        proteins, causes retention after maturation of the proteins by        passage into the Golgi apparatus. A discourse on the role of the        retention signal in protein maturation has been provided        by M. J. Lewis et al, (1992), cell, 68: 353-64.

More generally, the chimeric polypeptide sequences can comprise:

-   -   any sequence necessary for maturation of the protein in a        suitable cellular system;    -   any sequence necessary for recognition of a given cell type by        the chimeric molecule, thus enabling selectivity of action and        penetration into the cell cytoplasm.

The common factor between all chimeric sequences with structure B-X orB-X′ is that they contain the B fragment or a functional equivalentthereof.

The chimeric molecules of the invention enable X sequences or theexpression product of X′ to be routed in the ER. When X is bound to theB fragment, retrograde transport also occurs via the Golgi apparatus andprobably via the endosomes. Further, under certain conditions, themolecules of the invention can undergo maturation leading to a membranepresentation of certain epitopes contained in the chimeric polypeptidesequence.

The term “maturation” means any process which, from a given polypeptide,leads to the emergence of peptides which themselves can be presented ina cellular compartment including the cytoplasm. Maturation can occureither by enzymatic clipping in the endoplasmic reticulum, or bytransport into the cytoplasm in which the polypeptide is cleaved thenthe peptides obtained are again transported in the endoplasmicreticulum.

Molecules of the class I major histocompatibiliry complex (cl I MHC) canbecome charged with polypeptide molecules of interest X or X′ after suchcleavage and be presented on the cellular membranes.

When the chimeric molecule of the invention consists of coupling a Bfragment or its equivalent with a polynucleotide molecule or anexpression vector comprising a sequence the expression of which isdesired, after transcription in the nucleus then translation in thecytoplasm, the polypeptide which is synthesised can undergo the samesteps of cleavage, maturation and intracellular transport as thatdescribed above for a polypeptide chimeric sequence.

Chimeric polypeptide molecules in accordance with the invention canconstitute an active principle in a therapeutic composition forimmunotherapy by a mechanism which is close to biological processesregarding antigen presentation suitable for development of the immunereaction. The X fragment thus represents one or more epitopes for whichmembrane presentation is desired at the cell surface. The size of the Xfragment is limited only by the intracellular transit capacity of thechimeric molecules under consideration.

This approach can be envisaged both for an anti-infectious or ananti-cancer immunotherapy and for constituting an antigenic bait incertain autoimmune diseases.

Any type of antigen presented by cl I MHC is a good candidate forselecting simple or chimeric epitopes which form part of theconstructions of the invention. Examples which can be cited are:

a) Human Epitopes Derived from Melanoma Cell Proteins:

-   -   BAGE from tyrosinase (Boel, P et al (1995), Immunite 2,167-75);    -   GAGE from gp75 (Van den Eynde, B. et al (1995), J. Exp. Med.        182, 689-98;    -   tyrosinase (Brichard V. et al (1993), J. Exp. Med. 178,489-95);    -   pI5 from A/MART-1 melanoma (Coulie P. G. et al (1994), J. Exp.        Med. 180, 35-42; Kawakami Y. et al (1994), J. Exp. Med. 180,        347-52);    -   MAGE-1 and -3 from β-catenin (De Plaen E. et al (1994),        Immunogenetics 40, 369-9; Traversari C et al (1992), J. Exp.        Med. 176,1453-7.        b) Human Epitopes Derived from Virus Proteins Involved in Cancer        Development:    -   Peptides derived from E6 and E7 proteins of HPV 16        (Feltkamp M. C. et al (1993), Eur. J. ImmunoL 23, 2242-9;        Davis H. L. et al (1995), Hum. Gene Ther. 6, 1447-56);    -   Peptides derived from the Hbs protein of HBV (Rehermann B. et al        (1995), J. Exp. Med. 181, 1047-58);    -   Peptides derived from proteins from EBV (Murray R. J. et al        (1992), J. Exp. Med.    -   176,157-68);    -   Peptide derived from cytomegalovirus.    -   c) Human Epitopes Derived from Oncogenes:    -   p21ras (Peace D. J. (1993), J. Immunol 14, 110-4; Ciernik, I. F.        et at (1995)    -   Hybridoma 14, 139-42);    -   p53 (Gnjatic S. (1995), Eur. J Immunol. 25,1638-42).    -   d) Epitopes of Interest in Autoimmune Diseases:

These epitopes can be selected from those described by Chiez, R. M. etal (1994) in Immunol. Today 15, 155-60.

e) Epitopes of Interest in Infectious Diseases:

Examples of such epitopes which can be cited are those described byFurukawa K. et at (1994) in J. Clin. Invest. 94; 1830-9.

In the constructions of the invention, X or the expression product of X′can also represent a polypeptide sequence which can restore anintracellular transport function which has been perturbed by whatevercause. As an example, a biological molecule can be trapped in the ER dueto a modification by mutation, deletion or addition of a sequence,having the effect of blocking maturation or transit of that molecule.This is the case, for example, with mutated CFTR (Δ F508) 15 wherebinding to a chaperone molecule such as calnexin is modified such thatits release is prevented or retarded, thus preventing intracellulartransit. This mutation is the cause of cystic fibrosis. Introducing anon mutated replica into the endoplasmic reticulum could displaceN-glycosylated chains of the CFTR (Δ F508) glycoprotein from theinteraction site with calnexin, with the result that protein transportto the plasma membrane is renewed, and the epithelial cells of the lungfunction normally.

The invention also provides nucleic acid constructions, in particularDNA or cDNA comprising a sequence of nucleotides coding for the chimericprotein the structure and variations of which have been defined above.More particularly, the invention provides expression vectors or plasmidscarrying the above constructions and capable of being expressed inbacterial cultures. By way of example, the expression vector can be thepSU108 plasmid described by G. F. Su et al (1992), Infect. Immun. 60:3345-59.

More particularly, the invention provides constructions comprising:

-   -   the sequence coding for the B fragment;    -   a sequence coding for one or more polypeptides the expression of        which is desired. These may be epitopes the membrane expression        of which is desired at the cell surface; they may also be        polypeptides which can retain proteins in the Golgi apparatus;        finally, they may be polypeptides which can restore a disturbed        intracellular transport function.

The construction can also comprise any nucleic acid sequence coding fora polypeptide the presence of which enables proper intracellulartransport in cells intended to be treated by the molecules of theinvention. In particular, it may be:

-   -   a sequence coding for an N-glycosylation signal;    -   a sequence coding for the KDEL retention signal.

The polynucleotide construction of the invention is under the control ofa promoter, preferably a strong promoter which can produce the correctdegree of expression in bacteria into which it has been transfected.

The invention also provides transfected bacteria comprising theseconstructions, and capable of producing the chimeric polypeptides orproteins of the invention.

The host cells treated by the molecules of the invention also form partof the invention; they may be any type of cell, in particular:

-   -   those which can be treated in vivo such as immune system cells        which are active in triggering, cellular immunity, such as        dendritic cells, macrophages or B lymphocytes;    -   those which can be treated in situ such as epithelial cells for        use in restoring functions which have been altered either by a        genetic defect or by a metabolic perturbation;    -   cancer cells.

In general, the chimeric molecules of the invention allow a noveltherapeutic method to be postulated which can overcome the problemslinked to viral vectors or retroviral vectors which are normally used tointegrate and express exogenous molecules in animal cells. Thetherapeutic method which derives from the molecules of the inventionconsists of directly treating the cells of a patient, either ex vivo orby direct stereotaxic application with the chimeric polypeptidesequences, or by conventional mucosal treatment methods such asaerosols.

The invention concerns the use of chimeric polypeptide or polynucleotidesequences coding for the polypeptides of the invention in the productionof therapeutic compositions in which particular polypeptides areexpressed in the membranes of target cells. These polypeptides areadvantageously epitopes against which the development of animmunological reaction is desired which are then presented on thesurface of the immune system cells, in particular dendritic cells,macrophages or B lymphocytes. The B fragment of the Shiga toxin acts asan epitope vector enabling cells presenting antigens to be programmed.

The present invention concerns an immunotherapeutic method consisting ofincreasing cellular immunity as the result of the presence of anundesirable antigen in an organism, said method consisting of causingkey cells of the immune system, such as dendritic cells and macrophages,to express particular epitopes. The treatment method of the invention isaimed at triggering immunity to cellular and humoral mediation bycharging the cl I or cl II MHC molecules with the epitopes of interest,after restriction in the target cells. This leads to activation ofcytotoxic T cells against the antigen which it is desired to eliminate.

The epitopes presented through the constructions of the inventionoriginate from viral, parasitic or bacterial antigens or from any cell,organite, or micro-organism the elimination of which is desired, such ascancer cells or infected cells. The epitopes can also act as baitenabling “self” molecules recognised as foreign antigens in autoimmunediseases to be replaced by the epitopes of the invention, thus slowingdown or reducing the immune reaction.

Examples of these epitopes have been cited above in the description ofthe chimeric polypeptide sequences.

The invention also concerns the use of the chimeric molecules of theinvention in the manufacture of therapeutic compositions in which theparticular polypeptides which it is desired to express can restoreintracellular transit of a protein the altered structure of which leadsto it being trapped in the ER and to an expression deficit. This is thecase for membrane expression proteins which undergo intracellularmaturation, including glycosylations, sulphatations, folding etc.

A particular example is that of mutated CFTR (Δ F508) wherein theattachment of a chaperone molecule such as calnexin is modifiedfollowing modification of the protein; this leads to the molecule beingtrapped, causing cystic fibrosis, leading to a general insufficiency ofexocrin secretions, in particular in the pancreas and lungs.

The present invention concerns a therapeutic treatment method fordiseases having an origin in a fault in protein secretion; the methodconsists of directly administering the chimeric polypeptides oradministering the genetic information to the cells of patients in theform of plasmids carrying exogenic sequences coding for a peptide orpolypeptide which can restore the deficient cellular function.

This restoration can result either in supplementation of the deficientfunction by the polypeptide X or competition between the mutated proteinand the polypeptide synthesised from the exogenic sequence for bindingwith a specific molecule or receptor of the cellular machinery. Aparticular example is the treatment of the mutant cited above, causingcystic fibrosis, by administering a vector carrying a sequence codingfor the attachment site for the CFTR protein with its chaperone moleculeor by direct administration of the chimeric polypeptide.

The constructions of the invention endow the human or animal healthworld with a novel therapeutic means for treating diseases caused by adeficit in intracellular transit or for increasing or inducing amembrane presentation of a molecule, a polypeptide or an epitope ofinterest.

Further properties of the invention will become clear from the followingexamples and figures.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1)A: HeLa cells in which expression of the Gb₃ receptor has beeninhibited. The intemalised B fragment is not transported to the Golgiapparatus but is accumulated in the vesicular structures.

FIG. 1)B: Control HeLa cells in which the B fragment is transported tothe Golgi apparatus.

FIG. 2: Biochemical test showing the defect in B fragment transport.

FIG. 3: MHC class I restricted presentation of Shiga B-Mage 1 fusionproteins at peripheral blood monocytic cells (PBMC): role of the KDELsequence. The PBMC (5×10⁴) were primed overnight with either Mage 1peptide (1 μM) or Mart 1 peptide (1 μM) or Shiga B-Mage 1 fusionproteins (1 μM) with a sequence which is active (B-Mage 1-Glyc-KDEL) orinactive (B-Mage 1-Glyc-KDELGL) for recycling to the endoplasmicreticulum. After washing, 2×10⁴ cytotoxic T cells specific for the Mage1 epitope (clone 82/30) were incubated with PBMC cells primed for 24hours. The supernatants were then collected and tested for theproduction of γ interferon.

FIG. 4: MHC class I restricted presentation of the Shiga B-Mage 1 fusionprotein by different types of cells presenting antigens. Blymphoblastoid cells

dendritic cells (+) or clonal T cells

were primed with the soluble Shiga B-Mage 1 fusion protein, as for FIG.3. Presentation of Mage 1 peptides was tested using the 82/30 CTL line.

FIG. 5: Analysis of the specificity of the MHC class I restrictedpresentation of the Shiga B-Mage 1 fusion protein by lines of Blymphoblastoid cells. Cells from the BM21 (HLA-A1) or BV1 (HLA-A2) Blymphoblastoid line were primed overnight either with medium alone orwith Mage 1 or Mart 1 synthetic peptides (1 μM, or with ShigaB-Mage 1fusion protein (1 μM), or with Antp-Mage 1 fusion protein or with the Bfragment of wild-type Shiga toxin. After washing, specific cells of Mage1 TCL 82/30 (A) or Mart 1 CTL LB373 (B) were incubated for 24 hours withprimed B-EBV cells. The supernatants were then collected and tested forthe production of γ interferon.

I—CONSTRUCTION OF A RECOMBINANT CHIMERIC POLYNUCLEOTIDE AND THEPRODUCTION OF THE CORRESPONDING POLYPEPTIDE I—1) Construction of Plasmid

The X epitope selected was the MAGE epitope, present in cancer cells ofpatients with melanoma. The plasmid used was the pSU1O8 plasmiddescribed by Su et al, 1992, Infect. Immun. 60: 33-45, 3359.

The PCR primers used were as follows:

SEQ ID No. 1: 5′-ACTAGCTCTGAAAAGGATGAACTTTGAGAATTCTGACTCAGAATA GCTC-3′SEQ ID No. 2: 5′-CTTTTCAGAGCTAGTAGAATTAGGATGATAGCGGCCGCTACGAAAAATAACTTCGC-3′

The primers were used with specific primers from the ShigaAtpE (5′)vector

SEQ ID No. 3: 5′-CACTACTACGTTTTAAC-3′ SEQ ID No. 4:5′-CGGCGCAACTATCGG-3′to produce fragments which were cloned at the restriction sites SphI andSall of the SU108 plasmid.

Adapter fragments containing the glycosylation site and the KDELsequence composed of the oligonucleotides sulphate 1:((5′-phosphorylated; 5′-GGCCGCCATCCTAATTCTACTTCT-3′ (SEQ ID No. 5) andsulphate 2 (5′-CTCAGAAGTAGAATTAGGATGGC-3′ (SEQ ID No. 6)) or sulphate 3(5′-GAGTCTGAAAAAGATGAACTTTGATGAG-3′ (SEQ ID No. 7)) were ligaturedovernight at 16° C.

The resulting fragments were cloned at the NotI and EcoRI restrictionsites of pSU108 and containing the cDNA coding for B-Glyc-KDEL.

I-2) Purification of Proteins

The recombinant fragments were also purified using the techniquedescribed by Su et al, 1991, cited above. In brief, E. coli cellscontaining recombinant expression plasmids obtained from pSU108 werecultured overnight at 30° C. The culture was then diluted 5 times in LBsupplemented with 50 mg/ml of ampicillin, at 50° C. After incubation for4 hours at 42° C., the cells were thoroughly washed with 10 mM Tris/HCL,pH 8, incubated for 10 minutes in 10 mM Tris/Hcl, pH 8; 25% sucrose, 1mM EDTA, and finally rapidly re-suspended in a water-ice mixturecontaining 1 mM of PMSF and a protease inhibitor mixture (leupeptin,chymostatin, pepstatin, antipain and aprotinin). The final step led torupture of the periplasm. After clarification, the supernatant wascharged onto a QFF column (Pharmacia) and eluted with a linear gradientof NaCl in 20 mM Tris/HCl, pH 7.5. depending on the construction, the Bfragment was eluted between 120 mM and 400 mM. The fractions containingthe B fragment were then dialysed against 20 mM of Tris/HCl, pH 7.5, andre-charged onto a monoQ column (Pharmacia) and eluted in the same manneras before. The resulting proteins, estimated to have a degree of purityof 95% using polyacrylamide-SDS gel 15 electrophoresis, were then storedat −80° C. until use.

I—3) Induction of a CTL Response In Vitro

Dendritic cells (DC) were cultured using previously establishedprotocols (Romani et al, 1994). Briefly, PBMC were taken up intosuspension in Iscove medium and incubated for 2 h at 37° C. in 6-welltrays. Cells which had not adhered were removed and the remaining cellswere incubated at 37° C. in the presence of GM-CSF (800 U/ml) and IL-4(500 U/ml). After 5 days culture, the IL-1∝ and IFN-γ in respectiveconcentrations of 50 U/ml and 150 U/ml were added and incubation wascontinued at 30° C. for 24 h. The dendritic cells were then taken upinto suspension in Iscove medium in the presence of increasingconcentrations of B fragment coupled with the MAGE epitope and in thepresence of 3 μg/ml of human β2-microglobulin to improve the capacity ofthe cells to presentation of membrane epitopes. This mixture wasincubated at 30° C. for 4 hrs. DCs which had internalised the B fragmentcoupled to this epitope were irradiated at 5000 rad, assembled bycentrifuging, taken up into suspension, and mixed with CD8⁺ lymphocytes(prepared from PBMC). These DC, pulsed with an antigen, and the CD8⁺were then kept in co-culture in the presence of 5 ng/ml of IL-7.

After 10 days, responsive CD8⁺ lymphocytes were re-stimulated by freshlyprepared irradiated DCs which were then also incubated in the presenceof increasing concentrations of fragment B coupled to the same epitope.The co-culture of DC and responsive CD8⁺ lymphocytes was continued inthe presence of IL-2 and IL-7 in concentrations of 10 U/ml and 5 ng/mlrespectively. This re-stimulation protocol was repeated 3 times.

In order to measure the introduction of a CTL response, responsive CD8⁺lymphocytes pre-stimulated as described above were incubated in thepresence of cancer cells or cells infected with a virus. These cells,which expressed the selected epitope, were labelled with Na₂ ⁵¹CrO₄ thenbrought into contact with responsive CD8⁺ for 5 hours (Bakker et al,1994). The radioactivity released in the medium was then determined,enabling the cytotoxic activity of the pre-stimulated responsive CD8⁺lymphocytes to be quantified.

Results II—PRESENTATION OF THE MAGE 1 ANTIGEN BY PENA-EBV CELLS ANDDENDRITIC CELLS PULSED BY A B FRAGMENT OF THE SIGA TOXIN CARRYING THISANTIGEN II—1) Morphological Study of Intracellular Transport of a BFragment Carrying the MAGE-1 Epitope

We have demonstrated that it is possible to fuse a peptide sequence tothe carboxy-terminal end of the Shiga toxin B fragment while retainingintracellular routing of this protein towards the endoplasmic reticulum(ER). This demonstration was carried out by constructing chimericpolypeptides comprising the B fragment, the N-glycosylation site and theKDEL retention signal. As a control, the KDELGL retention signal wasused, namely the inactive version of the KDEL peptide, Misendock andRothman, 1995, J. Cell. Biol. 129: 309-319. Morphological andbiochemical studies have shown that the modified B fragment istransported from the plasmid membrane via the endosomes and the Golgiapparatus to the endoplasmic reticulum. This transport is inhibited byBFA (brefeldin A fungal metabolite) and reduced by nocodazole (amicrotubule depolymerisation agent).

These experiments clearly demonstrate that intracellular routing of thefusion protein towards the endoplasmic reticulum was retained. Toevaluate the potential of the B fragment as an epitope vector foranti-tumoral vaccination in vitro, the MAGE-1 epitope was added to theB-Glyc-KDEL fragment under the experimental conditions described above.The novel protein was designated B-MAGE-Glyc-KDEL. The B-MAGE-Glyc-KDELprotein was coupled with the fluorophore DTAF in order to follow itsintracellular transport by confocal microscopy. After internalistion,this protein was detectable in the Golgi apparatus and in the ER of HeLacells and Pena-EBV cells (a B lymphocyte line immortalised using theEpstein-Barr virus). These results confirm the original observationsconcerning the intracellular transport of the B fragment modified at itscarboxy-terminal end (described above) and affirm that certainpresenting cells of the hematopoietic line are capable of internalisingthe B fragment and transporting the protein to the ER.

We shall now describe these studies measuring the N-glycosylation of theB-MAGE-Glyc-KDEL protein. N-glycosylation is a modification which iscarried out specifically in the ER, and we have demonstrated above thata B fragment carrying a recognition site for N-glycosylation is in factglycosylated if it is transported to the ER.

II—2) Study of the Presentation of the MAGE-1 Antigen by Pena-EBV Cellsand Dendritic Cells Pulsed by the B-MAGE-Glvc-KDEL Protein

In order to evaluate the capacity of the fragment to act as an epitopevector, we used a cytotoxic T lymphocyte clone (CTL 82/30) specificallyrecognising the MAGE-1 epitope associated with MHC class I of cellspresenting the HLA-A1 haplotype. These CTL were kept in the presence ofPena-EBV cells or dendritic cells pulsed with the B-MAGE-Glyc-KDELprotein. If the MAGE-1 epitope is presented by presenting cells, the CTLwill be activated and will secrete γ interferon which (IFNγ) which isthen assayed.

The quantity of IFNγ secreted is proportional to the amplitude of thestimulation of the CTLs by the presenting cells.

Presenting cells (Pena-EBV and dendritic, 20000 cells per round bottommicrowell) were either fixed for 1 h with 4% PBS-paraformaldehyde, orwere not fixed. They were than washed twice with OptiMEM before beingincubated for 15 h with dilutions of the B-MAGE-Glyc-KDEL protein. Theprotein was tested at 4 dilutions, starting with a concentration of 10μmM final, and diluting 5 in 5 in the OptiMEM medium (medium withoutserum). After 15 h, the plates were washed twice by low speedcentrifuging. The CTL (CTL 82/30) were added in an amount of 5000 CTLper well in 100 μl of culture medium (ID-HS-AAG+25 U/ml of IL2). As apositive control, some CTL 82/30 were kept in the presence of line G43(a B lymphocyte line transfected with an expression plasmid of MAGE-1).After 24 h of incubation, the supernatants were harvested to determinethe quantity of IFNγ produced.

The results are shown in Table I.

TABLE I Concentration of B-MAGE-Glyc-KDEL Celltype 10 μM 2 μM 0.4 μM0.08 μM Den- Fixed 446 ± 293 821 ± 64 661 ± 18 312 ± 181 dritic Non 1557± 404  1315 ± 91  1231 ± 150 1174 + 478  fixed Pena- Fixed 70 ± 47  68 ±48  23 ± 32 4 ± 5 EBV Non 1966 ± 415  1960 ± 206 1544 + 42  853 ± 116fixed

The results are represented by the amount of EFNγ produced under eachset of conditions (average±standard deviation; n=3).

We can see that the dendritic cells and the Pena-EBV cells pulsed withthe B-MAGE-Glyc-KDEL protein were properly recognised by the CTL, evenat low concentrations of the protein. In contrast, the dendritic cellsand the Pena-EBV cells which had been previously fixed were notrecognised. It thus appears that endocytosis and processing of theB-MAGE-Glyc-KDEL protein had taken place. These encouraging results willnow be backed up by in vitro vaccination experiments.

III—IN VIVO ANTI-TUMORAL AND/OR ANTIVIRAL ACTIVITY TEST IN THE MOUSE

Mouse dendritic cells were prepared and marked with an antigen derivedfrom P21RAS, P53 or EP2/NER proteins to test the anti-tumoral activity,or HBV, EBV or HPV to test antiviral activity. This preparation ofdendritic cells was carried out using the protocol described in 1-4)above.

These dendritic cells were then introduced into the mouse.

The antiviral or anti-tumoral effect was observed by subsequenttreatment of these mice grafted with tumoral cells or virus expressingthis antigen.

Conclusion

The polypeptide sequences or polynucleotide sequences of the inventioncan thus advantageously constitute an active principle in apharmaceutical composition intended for the treatment of certain cancersor certain viral or bacterial infections, from the moment when aparticular epitope of said virus or said cancer cell will have beenintegrated into the recombinant nucleotide sequence, leading tosynthesis of a chimeric polypeptide which can be restricted by the MHCclass I and can be expressed on the membrane surface of immune systemcells.

IV—RESTORATION OF INTRACELLULAR TRANSPORT OF THE MUTATED PROTEIN CFTR(ΔF508) USING THE SHIGA TOXIN B FRAGMENT

The CFTR (cystic fibrosis transmembrane regulator) protein is a chlorinechannel of the plasmic membrane. In the large majority of patients withcystic fibrosis, the CFTR gene carries mutations. The mutation (ΔF508)which is the most frequently observed affects intracellular routing ofthe CFTR protein. In fact, the mutated protein CFTR(ΔF508), which isfunctional as regards its ionic channel activity, remains blocked at theendoplasmic reticulum, instead of being transported to the plasmicmembrane. Using the Shiga toxin B fragment, we have introduced a domainof the CFTR protein which is known to be the domain of interaction withthe calnexin protein (ER “chaperone”) into the endoplasmic reticulum.This domain is fused to the carboxy-terminal end of the B fragment. Wehave tested whether this chimeric protein can displace N-glycosylatedchains of the CFTR (ΔF508) glycoprotein from the interaction site withcalnexin, with the result that the CFTR (ΔF508) protein is no longerretained in the endoplasmic reticulum and can be transported to theplasmic membrane and thus function normally.

Firstly, we constructed a chimeric protein composed of a B fragment andthe interaction domain derived from the CFTR protein. A recycle signal(the KDEL peptide) was added to the carboxy-terminal end of this proteinto increase its retention in the endoplasmic reticulum. It was firstverified that the novel protein was also transported in the endoplasmicreticulum of target cells. Mobilisation of CFTR(ΔF508) was studied incells of a stable cell line, LLCPKI, transfected with the cDNA ofCFTR(ΔF508). This line was established by Mlle. M. A. Costa deBeauregard and M. D. Louvard (Institut Curie, Paris, CNRS UMR 144). TheCFTR (ΔF508) protein which was expressed in these cells was also endowedwith an epitope tag. It was thus possible to detect the arrival of theCFTR (ΔF508) protein in the plasmic membrane by immunofluorescence. Inthe absence of treatment, the plasmic membrane of LLCPKI cells of theline was depleted with specific CFTR (ΔF508) tags in the plasmicmembrane. While the results of these pilot experiments are promising, weare obliged to develop this approach within the context of cysticfibrosis therapy.

CONCLUSION

The experiment described above shows that the synthetic polypeptide inwhich X is constituted by an interaction domain between the

CFTR protein and calnexin can advantageously constitute the activeprinciple of a therapeutic composition intended to treat cysticfibrosis. In fact, competition between the mutated interaction domain inthe mutant and the fragment of synthetic polypeptide for the interactionwith calnexin can restore secretion of the mutated protein in thebronchia.

Antigenic Presentation Test:

Cells presenting the antigen (CMSP, B-EBV cells, T cells, dendriticcells) were incubated in 96-well microplates in a density of 10⁵ cellsper well and pulsed at 37° C. for 4 hours or 15 hours with the antigendissolved in 100 μ1 of Iscove medium. After incubation, the medium wasremoved and 20000 CTL cells were added to each well in 100 μl of CTLculture medium containing 25 U/ml of IL2. After 24 hours, 50 μl ofsupernatant was collected and the γ interferon was measured by an ELISA(Diaclone) test. In some experiments, the cells were fixed with 1%paraformaldehyde for 10 minutes at ambient temperature and washedthoroughly before transfer into the microplates.

1-18. (canceled)
 19. A method for antigenic presentation of an epitopeon a cell of the immune system, comprising: treating said cell with achimeric polypeptide of the formula:B-X wherein B represents the B fragment of Shiga toxin or a functionalequivalent thereof; and X represents one or more polypeptides, whereinsaid polypeptides are compatible with retrograde transport mediated by Bto ensure processing or correct addressing of X, such that said cellantigenically presents said epitope on said cell.
 20. The method ofclaim 19, wherein said cell is a dendritic cell or a macrophage.
 21. Themethod of claim 19, wherein said epitope is derived from viral antigens,parasitic antigens, bacterial antigens, cancer cell proteins, oncogenes,or cancerogenic virus proteins.
 22. A method for restoring intracellulartransit of a protein mutated at its site of attachment to a chaperonemolecule, comprising: administering to a patient a chimeric polypeptideof the formula:B-X wherein B represents the B fragment of Shiga toxin or a functionalequivalent thereof; and X is a polypeptide which can restoreintracellular transit, wherein said polypeptides are compatible withretrograde transport mediated by B to ensure processing or correctaddressing of X.
 23. The method of claim 22, wherein the mutated proteinis the CFTR protein responsible for cystic fibrosis,
 24. Apolynucleotide encoding a chimeric polypeptide of the formula:B-X wherein B represents the B fragment of Shiga toxin or a functionalequivalent thereof; and X represents one or more polypeptides, whereinsaid polypeptides are compatible with retrograde transport mediated by Bto ensure processing or correct addressing of X.
 25. A method forantigenic presentation of an epitope on a cell of the immune systemcomprising treating said cell with a chimeric molecule of claim 25, suchthat said cell antigenically presents said epitope on said cell.
 26. Themethod of claim 30, wherein said cell is a dendritic cell or amacrophage.
 27. The method of claim 30, wherein said epitope is derivedfrom viral antigens, parasitic antigens, bacterial antigens, cancer cellproteins, oncogenes, or cancerogenic virus proteins.